Molar Mass, Entanglement, and Associations of the Biofilm Polysaccharide of Staphylococcus epidermidis

Abstract

Biofilms are microbial communities that are characterized by the presence of a viscoelastic extracellular polymeric substance (EPS). Studies have shown that polysaccharides, along with proteins and DNA, are a major constituent of the EPS, and play a dominant role in mediating its microstructure and rheological properties. Here, we investigate the possibility of entanglements and associative complexes in solutions of extracellular polysaccharide intercellular adhesin (PIA) extracted from Staphylococcus epidermidis biofilms. We report that the weight average molar mass and radius of gyration of PIA isolates are 2.01 × 105 ± 1200 g/mol and 29.2 ± 1.2 nm respectively. The coil overlap concentration, c*, was thus determined to be (32 ± 4) × 10−4 g/mL. Measurements of the in situ concentration of PIA (cPIA,Biofilm) was found to be (10 ± 2) × 10−4 g/mL. Thus, cPIA,Biofilm < c* and the amount of PIA in the biofilm is too low to cause polymer chain entanglements. In the pH range 3.0 to 5.5, PIA was found to both self-associate and to form complexes with bovine serum albumin (BSA). By static light scattering, both self-association and complex formation with 0.5 %(w/v) BSA were found to occur at PIA concentrations of 0.30 × 10−4 g/mL and greater, which is about 30 times lower than the measured cPIA,Biofilm. These results suggest that the microscopic origin of EPS viscoelasticity is unlikely to be due to polysaccharide entanglements. Furthermore, the onset of self-association and protein complexation of PIA occurs at concentrations far lower than the native PIA concentration in biofilms. This finding therefore suggests a critical role for those two association mechanisms in mediating biofilm viscoelasticity.

Effects of Temperature on the Morphological, Polymeric, and Mechanical Properties ofStaphylococcus epidermidisBacterial Biofilms

“…Chemical analysis of these elution fractions, as discussed subsequently, suggest that from the left to right the first two peaks are from N -acetyl-glucosamine while the third and fourth peaks are due to both nucleic acid and proteins. This result is consistent with Ganesan et al 32 Moreover, the measured molar mass is consistent with the literature. Specifically, Ganesan et al 32 found that the second peak in the refractive index distribution displayed a mean molecular mass of (2.01 × 10 5 ) ± 1200 g/mol.…”

“…The Smith-Gilkerson assay was used to determine the presence of N -acetyl-glucosamine, a major component of the polysaccharide intercellular adhesion of the biofilm. 32,45 Similarly, a bicinchoninic acid assay (BCA) was used to determine the concentration of protein, and a microvolume spectrophotometric assay (Thermo Scientific NanoDrop 2000 spectrophotometer) was used to determine the concentrations of nucleic acid as well as protein. 46–48 …”

Role of Environmental and Antibiotic Stress on Staphylococcus epidermidis Biofilm Microstructure

“…43 In this region, the radial distribution function is related to the fractal dimension through the relationship, g ( r ) ~ r d f −3.44,45…”

Principles and application of laser light scattering (LLS) in characterization of the spatial configuration of microbial products in aqueous solution

“…As a result, the flocculation activity of the EPS from B. megaterium TF10 would be greatly enhanced after being dosed with substances that can bind the colloids to enhance the attraction, such as multivalent cations, and polymer electrolytes. A more negative A 2 (-3.14×10 -4 mol·L·g -2 ) was observed in the solution of extracellular polysaccharide intercellular adhesin (PIA) extracted from Staphylococcus epidermidis biofilms [43]. Consequently, the PIA would be expected to precipitate out of aqueous solution at high concentrations due to the attraction of the polymer chains.…”

“…Ions, especially the bridging cations, such as Ca 2+ , can mediate the aggregation of microbial products to increase the M w . For example, the extracellularly accumulated capsular assembly of glucuronoxylomannan molecules in Cryptococcus neoformans resulted from the attractive interactions between the carboxyl groups of glucuronic acid and divalent cations[42].Microbial products are always mixtures and their fractions can be characterized by the SLS technique using multi-angle laser light scattering (MALLS) detector coupled with separation techniques[38], such as size exclusion chromatography (SEC)[43,44], and asymmetrical flow field-flow fractionation (AF4)[45][46][47]. The determination of M w and R g requires that the concentration is simultaneously measured using another detector, typically based on refractive index (RI) or ultraviolet-visible (UV-vis).…”